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Development and application of a gen...

Marco, Maria Louise.

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Development and application of a genetic screen to identify Pseudomonas syringae pathovar syringae genes that are induced during colonization of leaf surfaces.

Record Type:	Language materials, printed : Monograph/item
Title/Author:	Development and application of a genetic screen to identify Pseudomonas syringae pathovar syringae genes that are induced during colonization of leaf surfaces./
Author:	Marco, Maria Louise.
Description:	170 p.
Notes:	Chair: Steven E. Lindow.
Contained By:	Dissertation Abstracts International63-09B.
Subject:	Agriculture, Plant Pathology. -
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3063471
ISBN:	0493823549

Development and application of a genetic screen to identify Pseudomonas syringae pathovar syringae genes that are induced during colonization of leaf surfaces.
Marco, Maria Louise.

Development and application of a genetic screen to identify Pseudomonas syringae pathovar syringae genes that are induced during colonization of leaf surfaces. - 170 p.

Chair: Steven E. Lindow.

Thesis (Ph.D.)--University of California, Berkeley, 2002.

We have identified a collection of <italic>Pseudomonas syringae</italic> pv. <italic>syringae</italic> strain B728a genes that are more highly expressed on leaves than in laboratory culture media. Identification of these genes was made possible by the application of a Habitat-Inducible Rescue of Survival (HIRS) system, a strategy that uses complementation of a conditionally lethal phenotype <italic>in vivo</italic> as a selection scheme. Our HIRS strategy to isolate plant-inducible genes of <italic>P. syringae</italic> was based on two <italic>P. syringae</italic> genes, <italic>metXW</italic> both required for methionine biosynthesis and survival on leaves during desiccation stress. Random fragments of <italic>P. syringae</italic> genomic DNA were inserted into a promoter trap plasmid (pTrap) such that they formed transcriptional fusions to a promoterless <italic>metXW</italic> operon and a <italic>gfp </italic> reporter gene. From a starting library consisting of 20,000 individual library clones, we isolated ≈130 clones that had a Met<super>+</super> phenotype on bean leaves but were Met<super>−</super> in a minimal medium. Transcriptional regulation of the plant-inducible loci contained in a subset of these clones was examined using appropriate fusions to promoterless <italic> gfp</italic> and <italic>inaZ</italic> reporter genes. While the transcriptional activities of the loci were usually too low to confer detectable levels of GFP fluorescence, ice nucleation activity measurements revealed that the putative plant-inducible genes were induced 10- to 100-fold on bean leaves compared to their expression levels in culture media. The DNA sequence of the plant-inducible loci was determined for 46 of <italic>metXW</italic> HIRS-selected clones. The loci were found to encode proteins involved in virulence, stress response, nutrient acquisition, membrane structure, chemotaxis, and other miscellaneous functions. We found that 52% of the loci contained novel ORFs and another subset harbored promoters located either in internal portions or on the non-coding strand of known genes. We performed a detailed analysis on two plant-inducible genes homologous to <italic>orf6</italic> and <italic>ssuE</italic>. Both of these genes were induced during dry conditions on leaves and their expression was probably highest in <italic>P. syringae</italic> cells located in protected sites on leaves. These sites are likely locations for <italic>P. syringae </italic> to express genes like <italic>orf6</italic>, which encodes a putative virulence factor associated with a <italic>hrp</italic>-encoded Type III secretory system, and <italic>ssuE</italic>, encoding an NAD(P)H-FMN reductase enzyme that would give <italic>P. syringae</italic> access to plant-derived sulfur sources. Furthermore, a <italic>P. syringae ssuE</italic> knockout mutant was unable to compete with wild-type cells for colonization in these sites on leaves during desiccation stress, suggesting the importance of nutritional adaptation. Thus, using the <italic>metXW</italic> HIRS system, we have isolated a variety of <italic>P. syringae</italic> genes specifically involved in colonization of leaf surfaces and, as a result, have contributed to an understanding of this important pathogen, ice nucleator, and epiphyte.

ISBN: 0493823549Subjects--Topical Terms:

1028950
Agriculture, Plant Pathology.

Development and application of a genetic screen to identify Pseudomonas syringae pathovar syringae genes that are induced during colonization of leaf surfaces.
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005 20110425
008 110425s2002 eng d
020 $a 0493823549
035 $a (UnM)AAI3063471
035 $a AAI3063471
040 $a UnM $c UnM
100 1 $a Marco, Maria Louise. $3 1251361
245 1 0 $a Development and application of a genetic screen to identify Pseudomonas syringae pathovar syringae genes that are induced during colonization of leaf surfaces.
300 $a 170 p.
500 $a Chair: Steven E. Lindow.
500 $a Source: Dissertation Abstracts International, Volume: 63-09, Section: B, page: 4041.
502 $a Thesis (Ph.D.)--University of California, Berkeley, 2002.
520 $a We have identified a collection of <italic>Pseudomonas syringae</italic> pv. <italic>syringae</italic> strain B728a genes that are more highly expressed on leaves than in laboratory culture media. Identification of these genes was made possible by the application of a Habitat-Inducible Rescue of Survival (HIRS) system, a strategy that uses complementation of a conditionally lethal phenotype <italic>in vivo</italic> as a selection scheme. Our HIRS strategy to isolate plant-inducible genes of <italic>P. syringae</italic> was based on two <italic>P. syringae</italic> genes, <italic>metXW</italic> both required for methionine biosynthesis and survival on leaves during desiccation stress. Random fragments of <italic>P. syringae</italic> genomic DNA were inserted into a promoter trap plasmid (pTrap) such that they formed transcriptional fusions to a promoterless <italic>metXW</italic> operon and a <italic>gfp </italic> reporter gene. From a starting library consisting of 20,000 individual library clones, we isolated ≈130 clones that had a Met<super>+</super> phenotype on bean leaves but were Met<super>−</super> in a minimal medium. Transcriptional regulation of the plant-inducible loci contained in a subset of these clones was examined using appropriate fusions to promoterless <italic> gfp</italic> and <italic>inaZ</italic> reporter genes. While the transcriptional activities of the loci were usually too low to confer detectable levels of GFP fluorescence, ice nucleation activity measurements revealed that the putative plant-inducible genes were induced 10- to 100-fold on bean leaves compared to their expression levels in culture media. The DNA sequence of the plant-inducible loci was determined for 46 of <italic>metXW</italic> HIRS-selected clones. The loci were found to encode proteins involved in virulence, stress response, nutrient acquisition, membrane structure, chemotaxis, and other miscellaneous functions. We found that 52% of the loci contained novel ORFs and another subset harbored promoters located either in internal portions or on the non-coding strand of known genes. We performed a detailed analysis on two plant-inducible genes homologous to <italic>orf6</italic> and <italic>ssuE</italic>. Both of these genes were induced during dry conditions on leaves and their expression was probably highest in <italic>P. syringae</italic> cells located in protected sites on leaves. These sites are likely locations for <italic>P. syringae </italic> to express genes like <italic>orf6</italic>, which encodes a putative virulence factor associated with a <italic>hrp</italic>-encoded Type III secretory system, and <italic>ssuE</italic>, encoding an NAD(P)H-FMN reductase enzyme that would give <italic>P. syringae</italic> access to plant-derived sulfur sources. Furthermore, a <italic>P. syringae ssuE</italic> knockout mutant was unable to compete with wild-type cells for colonization in these sites on leaves during desiccation stress, suggesting the importance of nutritional adaptation. Thus, using the <italic>metXW</italic> HIRS system, we have isolated a variety of <italic>P. syringae</italic> genes specifically involved in colonization of leaf surfaces and, as a result, have contributed to an understanding of this important pathogen, ice nucleator, and epiphyte.
590 $a School code: 0028.
650 4 $a Agriculture, Plant Pathology. $3 1028950
650 4 $a Biology, Ecology. $3 1017726
650 4 $a Biology, Microbiology. $3 1017734
690 $a 0329
690 $a 0410
690 $a 0480
710 2 0 $a University of California, Berkeley. $3 687832
773 0 $t Dissertation Abstracts International $g 63-09B.
790 $a 0028
790 1 0 $a Lindow, Steven E., $e advisor
791 $a Ph.D.
792 $a 2002
856 4 0 $u http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3063471